The HIV-1 glycoproteins function as homotrimers of gp120/gp41 heterodimers and represent a major target for vaccine development aimed at inducing neutralizing antibodies. HIV-1 virions carry very few Env spikes, 10 on average, thus theoretically limiting the neutralization target size relative to other enveloped virus. Stoichiometric investigations indicate that nAb neutralizes HIV-1 on the basis of individual Env trimers and binding a single antibody molecule is sufficient to neutralize the function of the whole Env trimer. In the Preliminary Results, we provide evidence that steric hindrance is sufficient for neutralization. Furthermore, we inserted the FLAG tag into the V4 region of HIV-1 strains YU2, JRFL and HXBc2, which have very different neutralization sensitivities. The V4 FLAG-tagged Envs of YU2, JRFL and HXBc2 were neutralized equivalently by the M2 anti-FLAG antibody, suggesting that antibody-binding affinity is a key determinant of neutralization efficiency. Taken in total, our studies support a simple model of antibody-mediated neutralization against HIV-1, in which the incidence of antibody binding to Env trimers determines neutralization. In this model, antibody-binding affinity is a key determinant and, once an antibody binds to an infectious HIV-1 Env spike, some level of neutralization must occur. In this proposal, we will develop this model by (1) defining the quantitative relationship between antibody-binding kinetics/affinity and neutralization efficiency and (2) determining if it generally applicable to different natural anti-HIV-1-Env nAbs. By defining the functional roles of antibody-binding affinity and target-specific/ non-target-specific steric hindrance in antibody neutralization against HIV-1, we will gain insights to better understand the mechanistic basis for HIV-1's resilience against antibody neutralization. By comparing divergent Envs from different enveloped viruses in these areas, the resulting information will help us to set priority for a prophylactic anti-HIV-1 vaccine by utilizing knowledge of vaccination successes against other viruses for the development of an anti-HIV-1 vaccine. Furthermore, these studies will contribute significantly to developing general principles of virology. Significance for Public Health: A major difficulty in developing antibody-based HIV-1 vaccines is that we do not yet know what is exactly required for the antibody responses to be effective. By studying the effect of antibody-binding affinity quantitatively and the role of target-specific and non-target-specific steric hindrance in antibody neutralization against HIV-1, the proposed works are designed to define such exact requirements that will be used as guiding posts for developing effective vaccine against HIV-1 infection, which is the ultimate weapon in combating the HIV-1/AIDS pandemic within this country and around the globe